Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis

Carbon nanotube tips for atomic force microscopy

The development of atomic force microscopy (AFM) over the past 20 years has had a major impact on materials science, surface science and various areas of biology, and it is now a routine imaging tool for the structural characterization of surfaces. The lateral resolution in AFM is governed by the shape of the tip and the geometry of the apex at the end of the tip. Conventional microfabrication routes result in pyramid-shaped tips, and the radius of curvature at the apex is typically less than 10 nm. As well as producing smaller tips, AFM researchers want to develop tips that last longer, provide faithful representations of complex surface topographies, and are mechanically non-invasive. Carbon nanotubes have demonstrated considerable potential as AFM tips but they are still not widely adopted. This review traces the history of carbon nanotube tips for AFM, the applications of these tips and research to improve their performance